Increased circulating ferritin and free iron has been found in a variety of disease states associated with thrombophilia (Kell, et al., Metallomics, DOI: 10.1039/c3mt00347g (2014). When iron is added to blood or plasma characteristic changes in thrombus formation are observed, which include fusion of fibrin polymers, matting, and even sheeting of fibrin (Kell, et al., Metallomics, DOI: 10.1039/c3mt00347g (2014); Lipinski, et al., Pol. Arch. Med. Wewn, 122:115-122 (2012); Lipinski, et al., Microsc. Res. Tech., 75:1185-1190 (2012;); Pretorius, et al., Microsc Res Tech, 76:268-271 (2013); Lipinski, et at., Curr. Neurovasc. Resi., 10:269-274 (2013); Pretorius, et al., Heart Lung Circ., 22:447-449 (2013)). These changes form a scanning electron micrographic (SEM) signature that has also been documented in thrombi obtained from patients with diseases involving chronic iron overload, such as diabetes mellitus and rheumatoid arthritis (Kell, et al., Metallomics, DOI: 10.1039/c3mt00347g (2014); Lipinski, et al., Pol. Arch. Med. Wewn, 122:115-122 (2012); Pretorius, et al., Blood Coagul. Fibrinolysis, 22:463-467 (2011); Pretorius, et al., Rheumatol. Int., 32:1611-1615 (2012)). With regard to mechanism, it has been posited that iron-derived hydroxyl radicals interact with fibrinogen, making it change as a substrate for thrombin, resulting in hypercoagulation and hypofibrinolysis (Kell, et al., Metallomics, DOI: 10.1039/c3mt00347g (2014); Lipinski, et al., Microsc. Res. Tech., 75:1185-1190 (2012;); Lipinski, et al., J Thromb. Thrombolysis, 29:296-298 (2010); Lipinski, et al., Hematology, 17:241-247 (2012)). Of interest, exposing plasma to iron chelators such as deferoxamine or antioxidants significantly attenuates SEM documented changes in clots subsequently exposed to exogenous iron (Pretorius, et al., Toxicol. Mech. Methods, 23:352-359 (2013)). Even more importantly, plasma obtained from patients with hemochromatosis or chronic hyperferritinemia demonstrated a SEM signature similar to that of iron exposure that can be attenuated following addition of deferoxamine (Pretorius, et al., PLoS ONE, 9:e85271 (2014)). In sum, while not directly demonstrated, it appeared that iron-fibrinogen interactions resulted in characteristic SEM determined morphology similar to that of diseases associated with chronic iron overload and thrombophilia.
However, the concept that iron modified fibrinogen via hydroxyl radical exposure is not supported by the observation that the procoagulant properties of fibrinogen are compromised by essentially all radical species tested by other investigators (Shacter, et al., Free Radic. Biol. Med., 18:815-821 (1995); Martinez, et al., Free Radic. Biol. Med., 65:411-418 (2013)). Indeed, given the typical range of soluble, nonenzymatic antioxidant concentrations present in human plasma (>1600 μM) (Yeum, et al., Arch. Biochem. Biophys., 430:97-103 (2004)), it is difficult to imagine that the addition of 6-30 μM ferric chloride (which markedly changes thrombus structure (Pretorius, et al., Toxicol. Mech. Methods, 23:352-359 (2013)) could selectively generate hydroxyl radicals that exclusively affect fibrinogen. Instead, it appeared that iron directly bound to fibrinogen in a recent study (Orino, Biometals, 26:789-794 (2013)). When this information is coupled with partial restoration of normal fibrin polymer architecture with iron chelation in clots derived from patients with hemochromatosis (Pretorius, et al., PLoS ONE, 9:e85271 (2014)), it seems more plausible that iron-fibrinogen binding and potential conformational change in the structure of fibrinogen may enhance it as a substrate for thrombin. This sort of iron-mediated phenomenon may be similar to that observed with enhancement of fibrinogen substrate characteristics following exposure to carbon monoxide (CO) (Nielsen, et al., Blood Coagul. Fibrinolysis, 22:443-447 (2011)). In short, the procoagulant effects of iron, a fibrinogen-binding atom, can be attenuated by introduction of deferoxamine prior to introduction of iron into plasma (Pretorius, et al., Toxicol. Mech. Methods, 23:352-359 (2013)), and the plasma of patients with chronic iron overload appear to be normalized following exposure to supra-pharmacological concentrations of deferoxamine (Pretorius, et al., PLoS ONE, 9:e85271 (2014)). The ability to prevent or reverse the effects of iron on coagulation with chelation speaks strongly for an important role of iron binding rather than radical damage as a major mechanism of iron-mediated enhancement of fibrinogen as a substrate for thrombin.
Chronic hemodialysis is also associated with significant thrombophilia (Nakamura et al., Nephron 58:201-204 (1991); Molino et al., Semin Nephrol 24:495-501 (2004); Zoccali et al., J Intern Med 254:132-139 (2003)). Hemodialysis patients have a high incidence of atherosclerotic disease, stroke, myocardial infarction and venous thromboembolism, and correlation of these disease states with specific hypercoagulable and hypofibrinolytic states have been made (Zoccali et al., J Intern Med 254:132-139 (2003); Undas et al., Nephrol Dial Transplant 23:2010-2015 (2008); Sharma et al., Eur Heart J 34:354-363 (2013)). Hemodialysis patients have been noted to have abnormal resistance to clot lysis, which has also correlated with the incidence of cardiovascular disease (Undas et al., Nephrol Dial Transplant 23 :2010-2015 (2008); Sharma et al., Eur Heart J 34:354-363 (2013); Segarra et al., J Am Soc Nephrol 12:1255-1263 (2001)).
Thrombophilia can also be caused by implanted devices. Left ventricular assist device (LVAD) implantation as bridge-to-transplantation (BTT) or destination therapy (DT) has become a mainstay of therapy for endstage congestive heart disease (Trivedi et al., Ann Thorac Surg 98:830-834 (2014); Donneyong et al., ASAIO J 60:294-299 (2014); Jorde et al., J Am Coll Cardiol 63:1751-1757 (2014)). Although the use of LVADs as BTT resulted in improved patient survival while waiting for transplantation, persistent thrombotic morbidity (e.g., stroke, pump thrombosis) still occurs in patients implanted with contemporary continuous flow LVADs despite optimized mechanical and medical therapy (Starling et al., N Eng J Med 370:33-40 (2014); Najjar et al., J Heart Lung Transplant 33:23-34 (2014); Trivedi et al., ASAIO J 59:380-383 (2013)). A concerted effort to identify biochemical markers that predict a tendency towards systemic hypercoagulability or impending pump thrombosis, such as circulating brain natriuretic peptide (BNP) and lactate dehydrogenase (LDH) activity have been proposed (Trivedi et al., ASAIO J 59:380-383 (2013)).
With regard to LDH as a measure of hemolysis during normal operation of various devices or after pump thrombosis, typical values are displayed in Table 1 derived from recent reports (Nielsen et al., Artif Organs 37:1008-1014 (2013); Smith et al., ASAIO J 59:93-95 (2013); Stepanenko et al., ASAIO J 57:382-387 (2011); Madden et al., ASAIO J 60:524-528 (2014); Bartoli et al., Thorac Cardiovasc Surg 62:414-418 (2014); Yoshioka et al., J Artif Organs 17(4):308-314 (2014); Whitson et al., Ann Thorac Surg 97:2097-2103 (2014)). There is significant device and center variability in low grade, hemolysis-generated LDH values, possibly indicative of differences in anticoagulation or blood flow characteristics dependent on surgical implantation and pump management (Inci et al., ASAIO J 58:373-381 (2012); Benk et al., Eur J Cardio-Thorac Surg 44:551-558 (2013)). This indolent but persistent process of device-mediated hemolysis is potentially biochemically important from a coagulation perspective, as free heme will upregulate heme oxygenase (Hmox, isoform 1 inducible, isoform 2 constitutive) activity and increase CO and iron release during heme catabolism (Owens Clin Biochem 43:1183-1188 (2010); Balla et al., Antioxid Redox Signal 9:2119-2137 (2007)).
TABLE 1LDH values associated with normal LVADoperation or during pump thrombosis.PatientReferenceDeviceNumberMean LDH (U/L)7HMII117009HVAD102344HMII837540 (normal)1490 (thrombosis)10HMII, HVAD, Jarvik 200045562-70411HMII, HVAD20279 (normal)2954 (thrombosis)12Jarvik 2000886013HMII193630HMII = HeartMate II; HVAD = HeartWare LVAD system; Jarvik 2000 = Jarvik 2000 LVAD (Jarvik Heart Inc., New York, NY, USA); normal = normal operation; thrombosis = pump thrombosis present. All normal values were obtained at least one month after pump placement.
Iron enhances coagulation and diminishes fibrinolysis via a recently reported mechanism (Nielsen et al., Blood Coagul Fibrinolysis 25(7):695-702 (2014)), and iron-enhanced coagulation has been documented in hemodialysis patients and in a patient with mitral valvular stenosis (Matika et al., ASAIO J 60(6):716-721 (2014); Thompson et al., J Thromb Thrombolysis epub ahead of print (2014)). Therefore, LVAD-associated upregulation of Hmox activity could result in iron-mediated hypercoagulation and hypofibrinolysis.
While the primary focus of investigations concerning device-associated thrombophilia have focused on patient-pump interactions, the contribution of comorbidities to hypercoagulability have not been fully appreciated. Patients implanted with LVADs have been noted to be afflicted with diabetes mellitus (DM) and obesity (Trivedi et al., Ann Thorac Surg 98:830-834 (2014); Donneyong et al., ASAIO J 60:294-299 (2014); Jorde et al., J Am Coll Cardiol 63:1751-1757 (2014); Starling et al., N Engl J Med 370:33-40 (2014); Najjar et al., J Heart Lung Transplant 33:23-34 (2014); Whitson et al., Ann Thorac Surg 97:2097-2103 (2014)) and both of these conditions have been associated with enhancement of Hmox activity (Bao et al., PLoS ONE 5:e12371 (2010); Nielsen et al., Blood Coagul Fibrinolysis 26(2):200-204 (2015)). Of interest, obstructive sleep apnea (OSA), a disorder often associated with obesity, has also been demonstrated to upregulate Hmox activity (Kobayashi et al., Chest 134:904-910 (2008)). Consequently, while patient-device interactions are important in the development of systemic hypercoagulability, comorbid conditions may further exacerbate Hmox-associated hypercoagulability via increased release of iron into the circulation.
Alzheimer's disease (AD) is also a risk factor for thrombophilia. AD patients have been documented to have abnormally increased activated Factor VII, von Willebrand factor and prothrombin 1+2 fragment in their circulation, the substrate and result of intravascular thrombin generation (Gupta et al., Int J Clin Pract 59(1):52-57 (2005)). Of interest, while patients with AD were noted to have fibrinogen concentrations that on average were not abnormally increased, another work noted that patients with mild cognitive impairment were at greater risk of more rapid advancement to AD if their circulating fibrinogen concentration was >300 mg/dl (Gupta et al., Int J Clin Pract 59(1):52-57 (2005); Xu et al., Int J Clin Pract 62(7):1070-1075 (2007)). Further, patients with AD have been noted to have plasma thrombus fibrin polymerization that is structurally similar to iron-exposed plasma, and AD patients with abnormally increased ferritin concentrations have red blood cell morphological changes, with increased membrane stiffness documented with atomic force microscopy (Lipinski et al., Front Hum Neurosci 7:735 (2013); Bester et al., Front Aging Neurosci 5:88 (2013)). In sum, there is biochemical and ultrastructural evidence of circulating abnormal intravascular thrombin generation, ridged red blood cells, and consequent abnormally appearing fibrin polymerization in thrombi obtained from patients with AD, all of which could contribute to spontaneous emboli.
Given the aforementioned morphological changes in fibrin in plasma obtained from AD patients, it is of particular interest that iron enhances plasmatic coagulation kinetics and modifies ultrastructure by modulating fibrinogen (Nielsen et al., Blood Coagul Fibrinolysis 25(7):695-702 (2014)). Iron decreases the onset time of coagulation, enhances the speed of clot formation, and acts as an antifibrinolytic agent when tissue-type plasminogen activator (tPA) is the fibrinolysin (Nielsen et al., Blood Coagul Fibrinolysis 25(7):695-702 (2014)). These facts are of importance in the setting of AD, as Hmox, the endogenous enzyme system responsible for heme catabolism, has been found to be enhanced in both the brain and circulation of AD patients (Barone et al., Neurobiol Dis 62:144-159 (2014); Barone et al., Free Radic Biol Med 52(11-12):2292-2301 (2012); Di Domenico et al., J Alzheimers Dis 32(2):277-289 (2012); Song et al., Exp Neurol 254:78-89 (2014)). Further, Hmox catabolism of heme results in the release of iron (Leffler et al., Am J Physiol Heart Circ Physiol 301(1):H1-H11 (2011)). However, inhibition of Hmox in a murine model of AD involving a double transgenic mouse (APPswe/PS1ΔE9) significantly diminished the behavioral deficits and neuropathological changes over time compared to mice not administered a Hmox inhibitor (Gupta et al., J Neurochem 131(6):778-790 (2014)). When all these data are considered as a whole, Hmox-enhanced coagulation could play a role in spontaneous thrombus formation not just in the peripheral circulation as documented by previous ultrasonic investigations but perhaps more regionally (and intensely) in the microcirculation of the brain itself.
Chronic migraine headache constitutes yet another risk factor for thrombophilia. Migraine headache afflicts and disables millions of people yearly worldwide (Bloudek et al., J Headache Pain 13:361-378 (2012); Leonardi et al., Neurol Sci 34:S117-S118 (2013); Mauser et al., Headache 54:1347-1357 (2014)). Specifically, nearly 12% of the population in the United States and close to 15% of citizens of several European countries experience episodic and chronic migraine (CM) headaches, with CM (>15 headaches per month for at least 3 months) occurring in 0.9%-2.2% internationally. Migraine headache is associated with significant economic loss secondary to lost productivity and medical expenses, and in terms of years living with disability, CM is the eighth most burdensome disease, and seventh among non-communicable diseases. The precise etiologies responsible for migraine are complex and poorly defined as recently reviewed, with brainstem dysfunction and dysfunction specifically of the locus coeruleus or periaqueductal grey matter likely playing a key role (Sprenger et al., BMC Med 7:71 (2009)). A variety of pharmacological treatments have attenuated severity and frequency of CM, varying from oral antiepileptic administration to botulinum toxin injection—yet the mechanisms by which many of these interventions attenuate migraine are poorly understood or not explained. In sum, CM is a significant condition with marked economic impact that remains only partially mechanistically characterized and incompletely attenuated with present-day therapies.
While a mechanism clearly responsible for neuroinflammation and central pain pathway dysregulation in CM has not yet been defined, there is a constellation of clinical and laboratory findings that may point the way to a hereto unappreciated contributor to CM. First, iron appears to accumulate in deep brain nuclei associated with central pain processing (e.g., putamen, globus pallidus, red nucleus) of patients with CM (Kruit et al., Cephalalgia 29:351-359 (2008); Kruit et al., Cephalalgia 30:129-136 (2009); Tepper et al., Headache 52:236-243 (2012)), and patients with hereditary hemochromatosis (C282Y/C282Y genotype) have a significantly increased incidence of headache that can be attenuated by therapeutic phlebotomy (Hagen et al., Ann Neurol 51:786-789 (2002); Stovner et al., Cephalalgia 22:317-319 (2002); Gaul et al., Headache 47:926-928 (2007)). The mechanism by which iron modulated headache was not determined in these works (Kruit et al., Cephalalgia 29:351-359 (2008); Kruit et al., Cephalalgia 30:129-136 (2009); Tepper et al., Headache 52:236-243 (2012); Hagen et al., Ann Neurol 51:786-789 (2002); Stovner et al., Cephalalgia 22:317-319 (2002); Gaul et al., Headache 47:926-928 (2007)).
A second line of evidence concerns the relationship of obesity with CM, wherein as body mass index (BMI, kg/m2) increased into the obese and morbidly obese value range, the incidence and severity of migraine also increased (Bigal et al., Neurology 66:545-550 (2006); Chai et al., Headache 54:459-471 (2014)).
Accordingly, weight loss, by either surgical or medical intervention, was associated with reduction in CM symptoms, clearly implicating obesity-mediated effects as key to CM (Novack et al., Cephalalgia 31:1336-1342 (2011); Verrotti et al., Pediatr Obes epub ahead of print (2014)). The third and last investigative line concerns the interaction of CM with coagulation. Migraineurs have over twice the risk of normal for thromboembolism, and while patent foramen ovale (PFO) may not be associated with migraine, migraineurs that have a PFO repaired are more likely to have headache relief than those that do not have repair (Schwaiger et al., Neurology 71:937-943 (2008); Garg et al., Circulation 121:1406-1412 (2010); Biasco et al., J Cardiol 64:390-394 (2014)).
Further, while migraineurs did not appear to have unusual hypercoagulable markers compared to headache-free controls, migraineurs administered vitamin K antagonists for conditions not related to headache improved headache symptoms, whereas patients administered clopidogrel as a prophylactic treatment did not realize significant relief from CM (Rajan et al., Clin Appl Thromb Hemost 20:851-856 (2014); Rahimtoola et al., Headache 41:768-773 (2001); Chambers et al., Cephalalgia 34:1163-1168 (2014)).
In sum, chronic migraineurs appear to have evidence of the involvement of iron in their pain, a contribution of obesity to their pain, and an ill-defined propensity towards plasmatic and not platelet-mediated hypercoagulability. The Hmox system, and in particular, Hmox upregulation, may be a common biochemical link between the aforementioned bodies of knowledge surrounding migraine. First, valproic acid, which has been used successfully to treat migraine, has been noted to be a Hmox inhibitor (Kwon et al., Neurochem Int 62:240-250 (2013)). Second, iron is a byproduct of Hmox catalysis of heme, and it enhances plasmatic coagulation via formation of carboxyhemefibrinogen (COHF) and iron-bound fibrinogen (IFIB) (Leffler et al., Am J Physiol Heart Circ Physiol 30: H1-H11 (2011); Nielsen et al., Blood Coagul Fibrinolysis 25:695-702 (2014)). Further, iron also diminishes fibrinolysis (Nielsen et al., Blood Coagul Fibrinolysis 25:695-702 (2014)).
Considered as a whole, as depicted in FIG. 12, it is conceivable that endogenous and exogenous CO and iron may interact with fibrinogen, enhance coagulation, modulate cerebral blood flow (e.g., microcirculatory occlusion-reperfusion) and contribute to migraine headache as either a source or amplifier of inflammatory central pain.
Given the likely importance of iron in the thrombophilic complications suffered by patients with several disease states (Kell, et al., Metallomics, DOI: 10.1039/c3mt00347g (2014)), it is an object of this invention to provide methods for diagnosing subjects having or suspected of having an iron-related disorder.
It is another object of the invention to provide methods for assessing the risk or likelihood that a subject has or will develop an iron-related disorder.
It is yet another object of the invention to provide methods for detecting iron-mediated plasmatic hypercoagulability and hypofibrinolysis in hemodialysis patients.
It is yet another object of the invention to provide methods for detecting iron-mediated plasmatic hypercoagulability and hypofibrinolysis in patients implanted with LVAD.
It is yet another object of the invention to provide methods for detecting iron-mediated plasmatic hypercoagulability and hypofibrinolysis in patients with Alzheimer's disease.
It is yet another object of the invention to provide methods for detecting iron-mediated plasmatic hypercoagulability and hypofibrinolysis in patients with chronic migraine.